Study On Pacemaker Channel Electrophysiological Properties

Cell is the basic unit of life. All organisms composed of cells, it is the basis oforganism growth and development. It’s also the basic unit of heredity. Overall, cell is thefundamental element of life. All animal cells are surrounded by a layer of film, which iscell membrane. It serves as a fence to keep intracellular materious and circumstancesstable. However, cell membrane is not absolutly sealed. Cells need to exchange materialsand energies with outside. Ion channels located in cell membrane perform this function.Ion channels can be classified into four types: ligand-gated channels, voltage-gatedchannels, intracellular signal-gated channels and mechanical sensitive channels.hyperpolarization-activated cyclic nucleotide-gated channel (pacemaker channel) is anon-selective cation channel which can be regulated by both voltage and cyclic adenosinemonophosphate (cAMP), the associated underlying current is Ih, Ihinvolved in regulatinga series of physiological processes, such as controling pacemaker activities in both heartand neurons, governing the resting membrane potential of some excitable cells, transferingnerve signals and signals in dendritic integration. Ihis regulated by the autonomic nervoussystem and neuroendocrine system and is affected by some pathological conditions. Itsdysfunction may induce many heart and mental diseases. Therefore, study Ihhas importantphysiological significances.At this present study, by using electrophysiological patch-clamp recording, whole cellCa2+imaging as well as a rat spared-nerve injury model, its regulation mechanisms, itsrole in neuropathic pain and its pharmacological properties are investigated.The main results are as follows:(1) The role of PKA signal pathway on regulating HCN channel activity. ProteinKinase A (PKA) is widely expressed in almost all types of cells, it involved in controllingand regulating a serial of pivotal physiological processes, while the effect of PKAinhibition on HCN channel activity is yet to be decided. To address this issue, we chosedKT5720, a selective PKA inhibitor. With using whole-cell voltage clamp, current clamp,single channel patch-clamp recording as well as Ca2+imaging, on freshly isolated DRGneurons. Our investigations revealed that PKA signal pathway plays a crucial role on regulating HCN channel activity, KT5720attenuated HCN channel activity. In presence of3μM KT5720, HCN channel currents density decreased. Its V1/2shifted tohyperpolarization direction, and the channel activation time constant significantlyincreased. Moreover, PKA inhibition with KT5720attenuated the action potentials (APs)and decreased intracellular Ca2+level, which therefore dampened DRG neuronsexcitability.(2) The role of HCN channel on regulating neuropathic pain. Neuropathic pain wascharacterized with high incidence and refractory, which often associated with nerve injury.It’s a major medical problem yet to be resolved. On the other hand, DRG neuron is theprimary sensory afferent, it’s essential for pain signal transportation. Hence, we choseDRG neuron as our study subject, on spared-nerve injury model, with using multipleelectrophysiological patch-clamp recording methods. We studied the underlyingmechanisms of HCN channel regulating neuropathic pain, unveiled that HCN channel is akey mediator on triggering and maintaining neuropathic pain. Under spared nerve injuryinduced pain condition, HCN channel activity in DRG neuron significantly increased, theectopic discharge frequency potentiated, and DRG morphology changed. Therefore, HCNchannel represents a novel potential target for the treatment of this disease.(3) The impact of ephedrine on HCN channel activity. Ephedrine is an adrenalagonist, which has the functions of strengthening heart contraction, enhancing cardiacoutput, potentiating heart rate and improving blood pressure as well. To investigate ifHCN channel involved in these regulations, we carried out experiments with conventionalwhole-cell patch-clamp recording method. We obtained HCN channel current Ihonneonatal rat hippocampal neurons and HEK cells. Our experimental results indicated thatIhattenuated by ephedrine. The likelihood mechanism is that HCN channel has somefeedback functions. When blood pressure or heart rate increases abnormally, HCN channelbeing able to partly block these momentums, and therefor maintains organisms’ health andlife safety.